U.S. patent application number 10/729854 was filed with the patent office on 2005-06-09 for micro/nano-fabricated glucose sensors using single-walled carbon nanotubes.
Invention is credited to Chung, JaeHyun, Lee, Junghoon, Lee, Kyong-Hoon.
Application Number | 20050124020 10/729854 |
Document ID | / |
Family ID | 34889602 |
Filed Date | 2005-06-09 |
United States Patent
Application |
20050124020 |
Kind Code |
A1 |
Lee, Junghoon ; et
al. |
June 9, 2005 |
Micro/nano-fabricated glucose sensors using single-walled carbon
nanotubes
Abstract
A novel glucose sensor utilizing hydrogen-specific gas sensing
capability of single walled carbon nanotubes assembled on
microelectrodes. Highly specific glucose sensing was demonstrated
using buffered sample solutions with clinically significant
concentrations. The approach enables a simple but powerful
bio-sensor reliably operating with a completely new principle, and
opens up novel device applications where functional nano-components
can be integrated into a bioMEMS device.
Inventors: |
Lee, Junghoon; (Seoul,
KR) ; Chung, JaeHyun; (Evanston, IL) ; Lee,
Kyong-Hoon; (Wilmette, IL) |
Correspondence
Address: |
REINHART BOERNER VAN DEUREN S.C.
ATTN: LINDA GABRIEL, DOCKET COORDINATOR
1000 NORTH WATER STREET
SUITE 2100
MILWAUKEE
WI
53202
US
|
Family ID: |
34889602 |
Appl. No.: |
10/729854 |
Filed: |
December 5, 2003 |
Current U.S.
Class: |
435/14 ;
435/287.1 |
Current CPC
Class: |
C12Q 1/54 20130101; C12Q
1/006 20130101; B82Y 30/00 20130101 |
Class at
Publication: |
435/014 ;
435/287.1 |
International
Class: |
C12Q 001/54; C12M
001/34 |
Claims
We claim:
1. A glucose sensor, comprising: an apparatus comprising a
plurality of metallic single-walled carbon nanotubes arranged and
configured to define at least one interstitial space for sorption
of hydrogen gas, at least one of said nanotubes positioned across
and in electrical contact with an electrode pair; and a glucose
oxidase component contacting said nanotubes.
2. The sensor of claim 1 wherein said glucose oxidase component
comprises a gas-permeable portion on said nanotubes.
3. A sensor of claim 2 wherein said glucose oxidase component
further comprises a liquid permeable portion.
4. The sensor of claim 1 further comprising a voltage source and a
current sufficient to at least partially reduce millimolar
concentrations of hydrogen peroxide.
5. The sensor of claim 4 further comprising an electrometer
responsive to hydrogen sorption on said nanotubes.
6. The sensor of claim 5 comprising an ohmmeter.
7. The sensor of claim 1 wherein said glucose oxidase is of
bacterial origin.
8. The sensor of claim 1 wherein said glucose oxidase component
comprises gluconic acid.
9. A method of sensing glucose, said method comprising: providing
an apparatus comprising a plurality of metallic single-walled
carbon nanotubes with a voltage potential thereacross, said
nanotubes arranged and configured to define at least one
interstitial space and contacting a glucose oxidase component;
introducing glucose to said glucose oxidase component; and
monitoring electrical response upon interstitial sorption of
hydrogen gas.
10. The method of claim 9 wherein said glucose is in a fluid medium
at a concentration greater than about 1 mM.
11. The method of claim 9 wherein said medium has a volume less
than about 10 .mu.L.
12. The method of claim 9 wherein said medium comprises a bodily
fluid.
13. The method of claim 9 wherein said glucose oxidase is of a
bacterial origin.
14. The method of claim 9 wherein said response is a change in
resistance of said nanotubes.
15. The method of claim 9 wherein glucose is oxidized to gluconic
acid.
16. A method of using metallic single-walled carbon nanotubes to
determine glucose concentration, said method comprising: providing
a plurality of metallic single-walled carbon nanotubes defining at
least one interstitial space, at least one of said nanotubes
positioned across an electrode pair, having an electrical
resistance, and a glucose oxidase component; introducing glucose to
said glucose oxidase component; applying a current across said
electrode pair at least partially sufficient to produce hydrogen
gas; and determining a change in said resistance upon said glucose
introduction.
17. The method of claim 16 wherein said glucose is in a fluid
medium at a concentration greater than about 1 mM.
18. The method of claim 17 wherein said medium comprises a bodily
fluid.
19. The method of claim 16 wherein said current is less than about
5 mA.
20. The method of claim 16 wherein said hydrogen gas is
proportional to said glucose introduced.
21. The method of claim 20 wherein said resistance change is
normalized.
22. The method of claim 21 wherein said normalized resistance is
compared to a standard scale of glucose concentrations versus
normalized resistance values.
Description
BACKGROUND OF THE INVENTION
[0001] Glucose sensing is a critical step towards the timely
diagnosis and treatment of diabetes, which is one of the major
diseases with great clinical attentions. There have been intensive
research and well-developed commercialized products to easily and
accurately monitor a blood glucose level. Non-invasive or minimally
invasive glucose monitoring has been considered as one of the vital
concerns for clinical applications, due to the need of frequent
monitoring and the inconveniences of blood sampling. Therefore, a
simpler and more reliable approach is desired, especially in terms
of the sensitivity to a very small-volume body fluid.
[0002] Carbon nanotube devices have been employed in a range of
chemical and biological sensor applications. See, U.S. Pat. No.
6,528,020, the entirety of which is incorporated herein by
reference. However, such devices are described in the '020 patent
only in the most general terms. Glucose sensing, for example, is
mentioned prospectively, without regard to device configuration or
mode of operation. The search for a reliable, accurate glucose
sensor continues to be a concern in the art. More recently and in
more detail, carbon nanotubes, have been proposed for potentially
high-sensitivity monitoring of glucose, but overall performance and
sufficient specificity for practical applications remain to be
proven. [K. Besteman, J. Lee, F. G. M. Wiertz, H. a. Heering, and
C. Dekker, "Enzyme-Coated Carbon Nanotubes as Single-Molecule
Biosensors," Nano lett., vol. 3, pp. 727-730, 2003.]
BRIEF DESCRIPTION OF THE DRAWINGS
[0003] FIG. 1. Conceptual illustration of chemical and physical
sorption of H.sub.2(.) on SWCNT bundles.
[0004] FIG. 2. A schematic diagram representing a glucose sensor
and carbon nanotube apparatus, in accordance with this
invention.
[0005] FIG. 3. A top view of a certain electrode configuration, as
can be used in conjunction with the sensor/apparatus of FIG. 2.
[0006] FIGS. 4A-G. With reference with FIG. 2, a schematic stepwise
illustration showing fabrication of a sensor/apparatus, in
accordance with this invention.
[0007] FIG. 5. Schematically, a detailed process for fabrication of
certain sensors/apparatus of this invention.
[0008] FIG. 6. Electrical characteristics of deposited SWCNTs (A)
I-V curve on SWCNTs (B) Time profile of SWCNTs during resistance
measurement.
[0009] FIG. 7. Response to H.sub.2O.sub.2 (SWCNTs spin-coated with
PDMS), in accordance with the methods and apparatus of this
invention.
[0010] FIG. 8. .DELTA.R/R on glucose concentration (at t=10
seconds).
[0011] FIG. 9. Response of SWCNTs on Buffer, H.sub.2SO.sub.4, and
H.sub.2.
[0012] FIG. 10. Response(.DELTA.R/R) to CO.sub.2 (1M).
[0013] FIG. 11. Response of fully H.sub.2 saturated and bare
SWCNTs.
SUMMARY OF THE INVENTION
[0014] In light of the foregoing, it is an object of the present
invention to provide various carbon nanotube sensors and/or devices
and method(s) for sensing glucose or determining glucose
concentration, thereby overcoming various deficiencies and
shortcomings of the prior art, including those outlined above. It
will be understood by those skilled in the art that one or more
aspects of this invention can meet certain objectives, while one or
more other aspects can meet certain other objectives. Each
objective may not apply equally, in all its respects, to every
aspect of this invention. As such, the following objects can be
viewed in the alternative with respect to any one aspect of this
invention.
[0015] It is an object of the present invention to provide an
assembly or configuration of single-walled carbon nanotubes
(SWCNTs) such that the interstitial spaces or sites thereof can be
used to sense or detect hydrogen gas in conjunction with the
presence of glucose.
[0016] It is an object of the present invention to provide a
chemical or biological system for use in conjunction with the
aforementioned nanotube assembly/configuration, such a system
producing directly or indirectly hydrogen gas in an amount related
to a particular analyte to be sensed, measured and/or monitored by
the nanotube assembly/configuration.
[0017] It is also an object of the present invention to provide a
sensor, apparatus and/or method for measuring or monitoring glucose
levels quickly at clinically-significant levels.
[0018] Other objects, features, benefits and advantages of the
present invention will be apparent from this summary and its
descriptions of various embodiments, and will be readily apparent
to those skilled in the art having knowledge of carbon nanotube
devices and assembly/fabrication techniques. Such objects,
features, benefits and advantages will be apparent from the above
as taken into conjunction with the accompanying examples, data,
figures and all reasonable inferences to be drawn therefrom, alone
or with consideration of the references incorporated herein.
[0019] The present invention relates, generally, to a glucose
sensor apparatus employing the hydrogen-specific gas sensing
capability of SWCNTs assembled on microelectrodes.
Dielectrophoresis can be used to collect and deposit metallic
SWCNTs in a bundle or an otherwise functionally-effective
configuration, the conductivity of which is selectively sensitive
to hydrogen. Hydrogen produced by glucose oxidation, and the
resulting electrometric change, is measured to quantify glucose
concentration Results show high-sensitivity monitoring of one sort
useful for non- or minimally invasive applications. For instance,
an extremely small amount of a body fluid can be analyzed rather
than whole blood in large quantity. Clinically important blood
gases, such as CO.sub.2 and O.sub.2, do not influence sensor
response nor does the presence of hydrogen ion, confirming high
specificity. The inventive sensor and/or method(s) also
demonstrated a quick response (about.ltoreq.10 sec) with simple
detection (e.g., resistance change), and easy fabrication.
[0020] Benefits associated with gas sensing by SWCNTs include room
temperature operation, simple configuration, quick response to
analytes, and very low detection limit. While the conductivity of
semiconducting SWCNTs changes in response to gases such as
nitrogen, ammonia, oxygen, etc., metallic SWCNTs are known to be
insensitive to such species. [P. G. Collins, K. Bradley, M.
Ishigami, and A. Zettle, "Extreme Oxygen Sensitivity of Electronic
Properties of Carbon Nanotubes," Science, vol. 287, pp. 1801-1804,
2000.]
[0021] It was recently predicted by quantum mechanical calculation
that gas molecules could be strongly adsorbed into inter-tube
interstitial spaces or sites of bundled or closely packed or
configured SWCNTs. [J. Zhao, A. Buldum, J. Han, and J. P. Lu, "Gas
molecule adsorption in carbon nanotubes and nanotube bundles,"
Nanotechnology, vol. 13, pp. 195-200, 2002.] The calculation also
predicted that, due to size limitations, only hydrogen molecules
would fit into the sites. Accordingly, providing a bundle of
metallic SWCNTs, a conductivity change would occur upon sorption of
hydrogen, but would not be significantly affected by other gas
species. (See FIG. 1)
[0022] When glucose is oxidized by a glucose oxidase (GOD),
H.sub.2O.sub.2 is produced as shown in equation (1). [See, e.g., A.
E. G. Cass, Biosensors: A Practical Approach, Oxford University
Press, New York, 1990.] Under a certain voltage potential,
H.sub.2O.sub.2 can be further electrolyzed to yield oxygen and
hydrogen. [See, Walter C. Schumb, Charles N. Satterfield, Ralph L.
Wentworth, Hydrogen Peroxide, American Chemical Society Monograph
Series, Chap. 8, Decomposition Processes, 1955, Reinhold Publishing
Corporation, New York, N.Y.] The conductivity will change due to
the adsorption of hydrogen in the interstitial sites of bundled
metallic SWCNTs. More specifically, the charge transport capability
of the SWCNTs will be reduced by the adsorption of hydrogen,
resulting in an increase in the electrical resistance.
Consequently, glucose concentration can be quantified by measuring
or monitoring an electrometric change, as the hydrogen gas produced
is proportional to the peroxide oxidation product and initial
glucose concentration.
.beta.-D-glucose+O.sub.2+H.sub.2O.fwdarw.Gluconic
acid+H.sub.2O.sub.2 (1)
[0023] Accordingly, the present invention is directed, in part, to
a glucose sensor. Such a sensor comprises an apparatus comprising a
plurality of metallic single-walled carbon nanotubes arranged and
configured to define at least one interstitial space or site for
sorption of hydrogen gas, with at least one of the nanotubes
positioned across and in electrical contact with an electrode pair;
and (2) a glucose oxidase component in contact with or proximate to
the nanotubes. As contemplated within the broader scope of this
invention, the oxidase component can contact the nanotubes, be
positioned thereon, thereabout or relative thereto sufficient to
function at least in part as described herein. In addition to an
oxidase enzyme, such a component can comprise a liquid-permeable
portion and a gas-permeable portion, the former for introduction of
a glucose-containing fluid medium to the component with the latter
to facilitate passage of a resulting oxidation product (e.g.,
hydrogen peroxide). Glucose oxidase is commercially available and
can be derived from a range of suitable biological sources.
[0024] The inventive sensor further comprises a voltage source
providing a current sufficient to at least partially reduce
physiologically or clinically-significant concentrations of
hydrogen peroxide produced upon introduction of glucose to the
oxidase component. An electrometer connected to the apparatus is
responsive to hydrogen sorption on the nanotubes. Typically, an
ohmmeter can be used; however, volt and amp meters can also be
utilized given the interrelationship of such current parameters.
Use of such a glucose sensor/apparatus can be evidenced by an
oxidase component gluconic acid as a residual byproduct of glucose
oxidation.
[0025] As inferred above, the present invention can also include a
method of sensing glucose. Such a method comprises (1) providing an
apparatus comprising a plurality of metallic single-walled carbon
nanotubes with a voltage potential thereacross, the nanotubes
arranged and configured to define at least one interstitial space
and in contact with a glucose oxidase component; (2) introducing
glucose to the oxidase component; and (3) monitoring electrical
response upon interstitial sorption of hydrogen gas. In certain
embodiments, such a response can comprise a change in conductive
resistance of the nanotube. Alternatively, voltage or current
change can be monitored upon hydrogen sorption, responsive to
glucose introduction. Such a method can be utilized to detect
glucose in a bodily fluid or diluted solution that has a sampled
bodily fluid at broad range of concentrations that include the
clinically significantly range of 1 mM to 10 mM. A very low
detection limit (0.028 mM.times.10 .mu.l) can be achieved when the
smallest measurable resistance variation (11 .mu..OMEGA./.OMEGA.)
is considered in the current experimental data. Likewise, the
methods of this invention are clinically useful in that regardless
of glucose concentration, fluid volumes less than about 10 .mu.L
can be used with good effect.
[0026] Alternatively, the present invention can be considered as a
method of using metallic single-walled carbon nanotubes to
determine glucose concentration. Such a method comprises (1)
providing a plurality of metallic single-walled carbon nanotubes
defining at least one interstitial space, at least one of the
nanotubes positioned across an electrode pair, having an electrical
resistance and in contact with a glucose oxidase component thereon;
(2) introducing glucose to the oxidase component; (3) applying a
current across the electrode pair at least sufficient to produce
hydrogen gas; and (4) determining a change in resistance upon
glucose introduction. At the low glucose concentrations and fluid
volumes typically utilized with this methodology, currents less
than about 5 mA can be applied with good effect. As demonstrated
below, the changes in resistance upon hydrogen sorption can be
normalized then compared to a scale of standard glucose
concentrations versus normalized resistance values, to determine a
subject glucose concentration.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0027] With reference to FIG. 2, in accordance with this invention,
glucose sensor 10 can comprise apparatus 12, voltage source 14 in
contact with electrodes 16 on substrate 18. As discussed elsewhere
herein, electrodes 16 can be provided in a pattern 20 conducive for
the deposition and placement of corresponding bundles or packed
configurations of carbon nanotubes 22. In certain embodiments,
electrodes 16 can comprise interdigitated comb pattern 20. (FIG. 3)
Common circuit components connect electrodes 16, source 14 and
electrometer (e.g., amp meter) 30. Voltage source 14 is
incorporated with electrometer 30 where circuit resistance (e.g.,
ohmmeter) is determined/monitored.
[0028] As discussed above, glucose oxidase component 24 contacts
nanotubes 22. With reference to FIG. 4, such a component can
comprise a gas permeable component 25 over nanotubes 22, electrodes
16 and, optionally, oxidized substrate 18' (FIG. 4A-D). Component
24 can further comprise liquid permeable portions 26 and 28
providing a matrix for glucose oxidase 27 (FIG. 4E-G). See, also,
Examples 1 and 2, below.
[0029] With reference to FIG. 3, electrodes 16 are designed and
patterned such that the gradient of the electric field produced
maximizes the number of attracted SWCNTs on both electrodes. Under
current conditions of the sort described herein, SWCNTs are
attracted to and deposited at the inner edge of each electrode, a
point of highest electric field gradient. When one or more SWCNTs
are so positioned, a higher electrical field gradient is generated
at the ends of each SWCNT, as the deposited resistance is much
smaller than the contact resistance, presumably due to the Schottky
barrier between the SWCNTs and the metal electrodes. Consequently,
a larger dielectrophoretic force is formed locally to attract more
SWCNTs, until the contact resistance becomes low enough to release
the concentrated electric field. Without limitation to any one
theory or mode of operation, the observed bundle or packed
configuration of SWCNTs is believed to be formed spontaneously in
this manner.
[0030] As described elsewhere herein, such a configuration of
metallic SWCNTs, deposited on the electrodes, are capable of
molecular hydrogen gas detection-believed due to size-exclusive
sorption facilitated by interstitial sites or spaces defined by the
nanotube configuration. Again, without limitation to any one theory
or mode of operation, it is believed that hydrogen gas is generated
by electrolysis of hydrogen peroxide, a glucose oxidation product.
Likewise, without limitation, it is believed a gas phase
electrolysis has at least a partial role in observed sensor
function. Accordingly, circuit connection between electrodes does
not necessarily involve SWCNTs of the bundled/packed configuration.
Numerous nanotubes are without electrode contact. While a large
electrical potential is present between the SWCNTs and the metal
electrodes (the Schottky barrier), the nanotubes have low
electrical conductivity. Gas-phase electrolysis (e.g., hydrogen
peroxide) is believed to occur in the electrical contact region,
with subsequent sorption (e.g., hydrogen) within interstitial
spaces of the non-electrode contacting nanotubes. Results observed
can be understood by analogy of the inter-electrode SWCNTs to a
non-aqueous electrolyte of an electrochemical cell.
EXAMPLES OF THE INVENTION
[0031] The following non-limiting examples and data illustrate
various aspects and features relating to the sensors, apparatus
and/or methods of the present invention, including the use of
single-walled carbon nanotubes for the detection or sensing of
hydrogen gas produced via glucose oxidation. In comparison with the
prior art, the present methods and sensors/apparatus provide
results and data which are surprising, unexpected and contrary to
the prior art. While the utility of this invention is illustrated
through use of several apparatus configurations and chemical
systems/components which can be used therewith, it will be
understood by those skilled in the art that comparable results are
obtainable with various other apparatus configurations and
components, as are commensurate with the scope of this
invention.
Example 1
[0032] It is known in the art that approximately 70% of grown
SWCNTs have semiconducting characteristics, while the remaining
approximate 30% have metallic characteristics. [R. Krupke, F.
Henmich, H. v. Lohneysen, and M. M. Kappes, "Separation of Metallic
from Semiconducting Single-Walled Carbon Nanotubes," Science, vol.
301, pp. 344-347, 2003.] According to the mechanism mentioned
previously, however, the present invention comprises sufficient
metallic SWCNTs in a nanotube population having a grouped or
bundled configuration to effectively sense hydrogen.
Dielectrophoresis can be used to collect SWCNTs with similar
electrical (e.g., metallic) properties. As demonstrated below, a
non-uniform electric field between a pair of electrodes induces an
attractive dielectrophoretic forces on particles, e.g., SWCNTs,
suspended in a suitable medium (e.g., dichlorobenzene solution). By
the dielectrophoretic force at a specific frequency band (e.g., at
1 MHz), metallic SWCNTs can be selectively collected and deposited
across a pair of electrodes. [R. Krupke, F. Henmich, H. v.
Lohneysen, and M. M. Kappes, "Separation of Metallic from
Semiconducting Single-Walled Carbon Nanotubes," Science, vol. 301,
pp. 344-347, 2003; H. A. Pohl, Dielectrophoresis, Cambridge Univ.
Press, Cambridge, 1978.] In contrast, a high-frequency electric
field produces a negative (repulsive) dielectrophoretic force on
semiconducting SWCNTs, and they are repelled from the electric
field. When a metallic SWCNT is deposited across electrodes by
dielectrophoresis, the electric field is deformed and concentrated
at both ends of the SWCNT, presumably due to Schottky barriers
between the SWCNT and metal electrodes. In this manner, many SWCNTs
can be deposited in densely packed bundles.
Example 2
[0033] As mentioned above, FIGS. 2-4 show schematically the
fabrication and structure of a glucose sensor and/or apparatus in
accordance with this invention. Further details relating to
substrate preparation and oxidation, electrode deposition and
patterning, and corresponding lithographic techniques are provided
in co-pending application Ser. No. 10/426,925, in particular
Examples 16-17 and FIGS. 20, 23-26 thereof, filed Apr. 30, 2003,
the entirety of which is incorporated herein by reference.
Likewise, as would be understood by those skilled in the art,
information relating to substrate, electrode and circuit
preparation is also provided in the aforementioned and incorporated
'020 patent, in particular columns 3-6 and by way of the figures
referenced therein. More specifically, with reference to FIG. 5,
SWCNTs (commercially, from HiPCo) individually separated and
dispersed in 1,2-dichlorobenzene by sonication (38) were deposited
on an oxidized Si substrate (32), as described above, on Cr/Au
(100/800 .ANG.) electrodes (34-40) by dielectrophoresis (electric
field: 1.4V/.mu.m@1 MHz). [P. Nikolaev, M. J. Bronikowski, R. K.
Bradley, F. Rohmund, D. T. Colbert, K. A. Smith, R. E. Smalley,
"Gas-phase catalytic growth of single-walled carbon nanotubes from
carbon monoxide," Chem. Phys. Lett., vol. 313, pp. 91-97, 1999.]
The sample was annealed (42) at 200.degree. C. to evaporate the
solvent (b.p.: 180.degree. C.). PDMS (polydimethylsiloxane, Sylgrad
184, Dow Corning Corp., Midland, Mich.) was prepared and
spin-coated (44-46) at 3000 rpm for 3 minutes as a gas-permeable
(non-permeable to liquid) membrane on the top of the
SWCNT-deposited electrodes. The PDMS membrane was baked (48) on a
hot plate (100.degree. C.) for an hour. A hydrogel (pHEMA,
2-hydroxyethyl methacrylate) was patterned (50) under a UV
radiation at 20 Watt/cm.sup.2 for 8 minutes. GOD from Aspergillus
niger (E.C.1.1.3.4, Sigma-Aldrich Corp., St. Louis, Mo.) was
immobilized (52) by physical adsorption for 30 minutes (1000U/10
.mu.L), and an additional hydrogel layer was patterned (54) to
entrap and maintain the enzyme. [M. Y. Arica and V. Hasirci,
"Immobilization of Glucose-Oxidase--a Comparison of Entrapment and
Covalent Bonding," J. Chem. Tech. Biotechnol, vol. 58, pp. 287-292,
1993.] The fabricated sensor was kept (56) in a phosphate buffer
(10 mM, pH 7.4) at 4.degree. C. for further use. In accordance with
this invention, various other substrate, electrode, oxidase and
gas-/liquid-permeable components--providing comparable
function--can be used herewith, as would be understood by those
skilled in the art made aware of this invention.
Example 3
[0034] Even though the deposited SWCNTs were barely visible under
scanning electron microscopy (SEM), the deposition result was
confirmed by a finite resistance after deposition. The electrodes
in FIG. 3 were designed and fabricated to have an interdigitated
comb (i.e., a so-called "Ramen") structure for a higher electric
field gradient and maximum concentration of deposited SWCNTs. As
expected, dielectrophoresis aggregated SWCNTs around the
electrodes, due to a concentrated electric field. The deposited
SWCNTs and porous structure of the GOD-immobilized hydrogel matrix
can be observed via SEM.
[0035] When a resistance was measured right after the SWCNT
deposition, the value was approximately .about.2 kOhm. When the
solvent was completely evaporated, a better electrical contact was
made, and the resistance reduced to a half, .about.1 kOhm. It was
confirmed that the deposited SWCNTs have metallic characteristics
as shown in FIG. 6A. During the measurement, the resistance
decreased due to a slight temperature increase by the current used
for measurement (FIG. 6B).
Example 4
[0036] The response pattern of a fabricated sensor to
H.sub.2O.sub.2 was evaluated [FIG. 7]. For purposes of this
experiment, the GOD reaction layer was not necessary, and the
device was coated only with PDMS. A droplet of H.sub.2O.sub.2
solution (10 .mu.L) was placed above the SWCNT-deposited
electrodes, and a resistance was monitored. Solutions of different
concentrations were used including DI water and a series of diluted
H.sub.2O.sub.2 (2, 4, 6, 8, 10 mM). After each droplet was applied,
the resistance increased and reached a steady-state value. The
normalized resistance change (.DELTA.R/R) for the initial 10
seconds was related to the concentration of H.sub.2O.sub.2. The
increase of the resistance reached a maximum at 6 mM and began to
decrease at 8 mM. This observation is believed due to the
self-decomposition of hydrogen peroxide at higher concentrations.
The decomposition leads to generation of proton ions that caused a
response shift in the opposite direction (vide infra), a condition
resolved by controlling the density of deposited SWCNTs and the
concentration of immobilized enzyme for a device with reaction
layers.
Example 5
[0037] Devices with whole reaction and diffusion layers integrated
were used for testing glucose solutions at various concentrations.
The normalized resistance change was measured at 10 seconds after
the glucose solution (1, 5 and 10 mM) was placed on the device.
FIG. 8 illustrates that the resistance increased according to the
increase of glucose concentration. Concentrations much lower than 1
mM, can be readily measured as shown in the trend of the graph In
principle, a very low detection limit (less than about 0.028
mM.times.10 .mu.l) can be achieved as smaller measurable resistance
variations (e.g., less than about 11 .mu..OMEGA./.OMEGA.) are
considered.
Example 6
[0038] The sensor/apparatus responded insignificantly to other
gases or chemical species produced as a result of the GOD reaction
or as can exist in a blood sample (e.g., hydrogen ion, O.sub.2 and
CO.sub.2, FIG. 9). An extremely high concentration (1M) of
bicarbonate ion (HCO.sub.3.sup.-), carrying CO.sub.2 and hydrogen
ion in blood, did not cause any response change (FIG. 10),
indicating a physiological increase in pCO.sub.2 or pH appears not
to influence the sensor performance. Protons caused the signal to
decrease only at an abnormally high concentration (e.g.,
H.sub.2SO.sub.4, 12M). For the phosphate buffer (pH 7.4), the
resistance ratio increased a little possibly owing to diffusion of
H.sub.2 molecules. When the device was placed in a chamber with
H.sub.2 only, the resistance drastically increased as expected.
EXAMPLE 7
[0039] The effect of exposure to air (i.e., oxygen and nitrogen)
was also tested. FIG. 11 shows that an abrupt increase or decrease
of air concentrations using a repeated evacuation process had no
effect on the sensor signal. A fully H.sub.2 saturated SWCNTs that
had been placed in a buffer solution for a few hours showed a
signal decrease due to the time-dependent depletion of hydrogen
molecules from the interstitial sites when subsequently placed in
air or in vacuum However, the SWCNTs in this case did not respond
to air concentration. These results confirm that neither oxygen nor
nitrogen affect the electrometric response of or charge transfer
process associated with a metallic SWCNT configuration of this
invention.
[0040] While the principles of this invention have been described
in connection with specific embodiments, it should be understood
clearly that these descriptions are added only by way of example
and are not intended to limit, in any way, the scope of this
invention. For instance, the present invention can be applied more
specifically to a range of other possible analytes and/or enzymatic
systems producing, directly or indirectly, hydrogen gas or hydrogen
peroxide. Regarding the latter, however found or generated, it was
often important to quantify its concentration at low levels. The
apparatus and method(s) of this invention may be used in
conjunction with bioassays as an alternative to the hydrogen
peroxidase systems currently employed. Likewise, as would be
understood by those skilled in the art, the present invention can
be utilized in conjunction with the retention or storage of
hydrogen, for subsequent use or application. Other advantages,
features and benefits of this invention will become apparent from
the claims hereinafter, as would be understood by those skilled in
the art.
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